Physical techniques of membrane studies -- Study of membrane active peptides in bilayers

Abstract

Biological functions of cell membranes are mediated by specific proteins. These proteins assume their active forms only in the membrane environment. We review two methods of direct studies of the conformations of molecules embedded in bilayer membranes. We prepared membranes containing proteins into aligned multilayers. Such multilayers possess one-dimensional structural order and preserve the orientational order of proteins relative to the plane of membrane. Membrane proteins often contain helical sections. The orientation of these helical sections can be determined by the method of oriented circular dichroism. The one-dimensional structures can be determined by X-ray diffraction off aligned multilayers. In particular, the perpendicular postions of the label atoms in membrane proteins can be determined to within a few tenths of angstrom. These methods are illustrated with the applications to the structural problems of two membrane active peptides: alamethicin and gramicidin. Oriented circular dichroism showed that alamethicin switchs between the perpendicular orientation and the parallel orientation to the plane of membrane as we vary the chemical potential of water and the peptide-lipid ratio. The locations of the ion binding sites in the gramicidin channel were determined by two different X-ray diffraction methods. In one, the diffraction patterns of membranes containing gramicidin and ions (Tl +, K +, Ba ++, Mg ++ or without ions) were measured and the ion binding sites were revealed by the difference electron density profiles. In another, the ion binding sites were found by anomalous multiple-wavelength diffraction.